Pancreatic cancer is considered as a chemoresistant tumor because of its
abundant cancer stroma which contains cancer-associated fibroblasts (CAFs).
Nab-paclitaxel (Nab-PTX, cytoskeletal anticancer drug) is expected as a key
drug for pancreatic cancer. The patients of pancreatic cancer treated with
Nab-Paclitaxel show remarkable tumor reduction in size. We analyzed total 20
surgically resected cases of pancreatic ductal adenocarcinoma; ten cases
treated with Nab-PTX before surgical operation (treated group) and ten cases
without neoadjuvant chemotherapy (untreated group). The treated group showed
high density of aniline blue (AnB) positive collagen bundles in the stroma and
low density of α-smooth muscle actin (α-SMA) positive CAFs. On the other hand,
the untreated group exhibited low density of AnB positive collagen bundles and
high density of CAFs. We speculated that the Nab-PTX neoadjuvant chemotherapy
plays in roles of stromal collagenous fibrosis and inactivation of CAFs.

Pancreatic cancer
shows one of the poorest patient prognoses among all types of human cancer [1].
The majority pancreatic cancer is invasive ductal carcinoma, i.e., pancreatic
ductal adenocarcinoma [2-4]. Histopathologically, pancreatic cancer tissues have
abundant cancer stroma and many cancer-associated fibroblasts (CAFs) which are
considered to be one of the major factors contributing to the poor prognosis of
pancreatic cancer [5,6]. Previously the pancreatic cancers were chemoresistance
[7], but the recent chemotherapy has been improved the patient prognosis
gradually. Nab-Paclitaxel (Nab-PTX) has been expected as a key drug for the
pancreatic cancer treatment. The patients with pancreatic cancer performed
neoadjuvant chemotherapy with Nab-PTX frequently showed remarkable tumor
reduction in size. Paclitaxel (PTX) is one of cytoskeletal drugs, and its
derivative Nab-PTX is developed to improve solubility binding to human serum
albumin. The main mechanism of PTX is to inhibit the depolymerization of microtubules.
A few studies have reported Nab-PTX controls tumor by reducing CAFs [8,9].
However, the anticancer mechanisms of Nab-PTX are still incompletely
understood.

In this study we
investigated why Nab-PTX has prominent anticancer effects, analyzing histopathological
specimens of pancreatic cancer stroma which were performed neoadjuvant
chemotherapy with Nab-PTX.

MATERIALS AND METHODS

We investigated total 20 surgically resected
cases of pancreatic ductal adenocarcinomas; ten cases treated with Nab-PTX
before surgical operation (treated group) and ten cases without neoadjuvant
chemotherapy (untreated group). Numbers of the cases were limited because the
Nab-PTX treatment has recently established as the neoadjuvant chemotherapy for
pancreatic cancer.

We evaluated the cancer stromal phenotypes
using Heidenhain’s AZAN trichrome stain (AZAN stain) [10,11] and
immunohistochemical α-smooth muscle actin (α-SMA) stain. Collagen bundles in
the cancer stroma are stained with aniline blue (AnB) of AZAN stain, while CAFs
in the cancer stroma are
immunohistochemically positive for α-SMA. We

used imaging software
“Image J” to analyze fibrosis of cancer stroma, i.e., quantification of
collagen bundles (AnB) and CAFs (α-SMA). We selected three invasive foci which
represented the most intense fibrosis, and took photographs with x40 objective
lens for all cases. Then, we analyzed binarized/extracted images of collagen
bundles (AnB) and CAFs (α-SMA) using the software. The images were represented
as pixels on the computer screen. We calculated the average of three areas for
all the cases.

RESULTS

Pancreatic ductal
adenocarcinoma was characterized by an invasive growth pattern with abundant
fibrous stroma in both treated/untreated groups (Figure 1). However, the treated/untreated groups revealed
different distribution pattern of AnB positive collagen bundles and α-SMA
positive CAFs. The treated group showed the very high density of collagen
bundles and the low density of CAFs. On the other hand, the untreated group
exhibited the low density of collagen bundles and high density of CAFs.

In the present study,
we demonstrated the Nab-PTX neoadjuvant chemotherapy induced the unique fibrous
stroma of pancreatic ductal adenocarcinoma, i.e., dense AnB positive fibrous
stroma with limited numbers of CAFs. We think the dense collagenous fibrosis in
the stroma contributes to tumor shrinkage in an anticancer effective manner. We
have speculated that the pancreatic ductal adenocarcinomas in the untreated
group continuously induced active CAFs in the stroma, and made use of the CAFs
for the chemoresistance [5,6]. However, the treated group with Nab-PTX rapidly
induced AnB positive collagen bundles in the stroma, and then decreased numbers
of CAFs.

Our previous studies
reported that the transcriptional factors associated epithelial-mesenchymal
transition (EMT) contributed pancreatic cancer invasiveness/aggressiveness, and
were thought to be related with CAFs [12,13]. The Nab-PTX neoadjuvant
chemotherapy plays in roles of stromal collagenous fibrosis and inactivation of
CAFs. However, details of molecular mechanisms of Nab-PTX are still unknown. We
try to analyze the molecular mechanisms of collagenous fibrosis as the Nab-PTX
anticancer effects in the near future.